1. Field of the Invention
The present invention relates to improvements in methods and apparatus for sensing and characterizing small particles, such as blood cells or ceramic powders, suspended in a liquid medium having an electrical impedance per unit volume which differs from that of the particles. More particularly, it relates to improvements in methods and apparatus for sensing and characterizing such particles by the Coulter principle.
2. Discussion of the Prior Art
U.S. Pat. No. 2,656,508 to Wallace H. Coulter discloses a seminal method for sensing particles suspended in a liquid medium. An exemplary apparatus for implamenting such method comprises a dual-compartment dielectric vessel which defines first and second compartments separated by a dielectric wall. Each of the compartments is adapted to contain, and is filled with, a liquid medium. The particles to be sensed and characterized are suspended at an appropriate concentration in the liquid medium and introduced into one compartment through a suitable inlet port formed therein. The separating wall is provided with a relatively large opening which is sealed by a thin wafer made of a homogeneous dielectric material. A small through-hole formed in the wafer provides a conduit, which constitutes the only operative connection between the two compartments. An appropriate vacuum applied to an outlet port suitably formed in the second compartment causes the particle suspension to flow from the first compartment to the second compartment through said conduit, discussed in detail below. Each particle in the suspension displaces its own volume of the particle-suspending liquid, and the conduit provides a consistent reference volume against which that displaced volume may be compared. If the dimensions of the conduit and the concentration of particles in the suspension are appropriately selected, particles can be made to transit the conduit more or less individually. The conduit thus functions as a miniature volumeter, capable under suitable conditions of making sensible the liquid displaced by individual microscopic particles.
To enable convenient sensing of the liquid displacement occasioned by particles transiting the conduit, the particle-suspending liquid is made to have an electrical impedance per unit volume which differs from that of the particles. The contrast in electrical impedance between particle and suspending liquid thus converts the volume of displaced liquid into a proportional change in the electrical impedance of the liquid column filling the conduit. An excitation electrode is positioned in each of the two compartments and operatively connected to a source of electrical current, whereby a nominal electrical current (the excitation current) is caused to flow through the conduit simultaneously with the particle suspension. Consequently, passage of a particle through the conduit produces a pulsation in the current flowing through the conduit which is proportional to the volume of liquid displaced by the particle. An extensive art has developed whereby such particle pulsations may be sensed and monitored to provide particle-characterization information. This art has taken two forms, the first based on the original two-terminal sensing approach described in the '508 patent and the second based on four-terminal, potential-sensing approaches. The second form of Coulter art evolved from the first and shares similar limitations; both forms will be discussed.
In the '508 patent the excitation current is applied from a voltage source through the two electrodes immersed in the suspending liquid of the two compartments interconnected by the conduit. An AC-coupled sensing circuit, also operatively connected to the excitation electrodes, operates to sense the pulsations in current between these electrodes. Thus, as individual particles pass through the conduit, said sensing circuit produces an electrical signal pulse having an amplitude which is characteristic of the particle volume. Additional circuits further process the particle signal pulses to provide a count of particles exceeding some particular volumetric threshold or, via the elegant positive-displacement metering system disclosed in U.S. Pat. No. 2,869,078 to Wallace H. Coulter and Joseph R. Coulter, Jr., the particle concentration. The volumetric distribution of the particles may be conveniently characterized by causing the current source to provide a constant current and analyzing the particle pulses with multiple-thresholding sizing circuitry as described in U.S. Pat. No. 3,259,842 to Wallace H. Coulter et al. Alternatively, if the current source is caused to provide combinations of electrode excitation, including at least one source of high-frequency alternating current as discussed in U.S. Pat. Nos. 3,502,973 and 3,502,974 to Wallace H. Coulter and W. R. Hogg, an apparent volume reflecting the internal composition of certain particles may be similarly characterized. Such characterization results are displayed or recorded by appropriate devices. This method of sensing and characterizing particles, by suspending them in a liquid medium having an electrical impedance per unit volume which differs from that of the particles and passing the resulting particle suspension through a constricting conduit while monitoring the electrical current flow through the conduit, has become known as the Coulter principle. Because of their simplicity, the two-terminal sensing methods were the only ones in use for many years and still see exclusive use in commercially available apparatus incorporating the Coulter principle.
Central to the Coulter principle is the volumeter conduit which enables electrical sensing of particle characteristics by constricting both the electric and hydrodynamic fields established in the dual-compartment vessel. Due to their excellent dielectric and mechanical properties, ruby or sapphire jewels developed as antifriction bearings for precision mechanical devices were indicated for use as conduit wafers in U.S. Pat. Nos. 2,985,830 and 3,122,431 to Wallace H. Coulter et al. As shown in the enlarged longitudinal section of a traditional Coulter conduit wafer W in FIG. 1, a Coulter volumeter conduit comprises a continuous surface or wall 30 of length L which defines a right cylindrical opening of circular cross-section and diameter D through a homogeneous dielectric material of thickness L. (Conduit wafer W is often called an "aperture wafer", and volumeter conduit 10 in conduit wafer W is commonly referred to as a "Coulter aperture".) Due to material homogeneity, the electrical resistivity of conduit wall 30 surrounding the flows of particle suspension and current through the conduit is substantially axisymmetric and uniform in any longitudinal conduit section.
In practice, FIG. 1 conduit diameter D must approach twice the maximum particle diameter to minimize risk of clogging, and conduit length L is usually made as short as possible to minimize coincidence artifacts due to two or more particles simultaneously transiting the conduit. For many medical and scientific applications, conduit diameter D ranges between approximately 0.030 mm and 0.200 mm, and the conduit length-to-diameter ratio UD ranges between 0.75 to 1.2. With these small conduits, physical clogging may limit application, especially to samples of biological origin, while their limited dynamic range in particle size may limit application where polydisperse industrial samples are involved. Critical applications can benefit from splitting such samples through a plurality of conduits and appropriately processing the multiple data streams. Parallel-conduit systems typically comprise a single entry compartment containing a first electrode, but provide each conduit with an electrically isolated exit compartment containing an individual second electrode, with all conduits being simultaneously transited by parcel streams split from the sample suspension in the entry compartment. As taught by Wallace H. Coulter and W. R. Hogg, the several conduits may be either of identical geometry (U.S. Pat. No. 3,444,483) or of dissimilar geometry (U.S. Pat. No. 3,603,875). Each conduit independently generates pulse data for the particles in its portion of the split sample. In the first case voting logic may be used to eliminate from the redundant particle data any data originating in a clogged conduit, while in the second, pulse-analytic circuitry may be used to process the dissimilar data into a composite volumetric distribution for polydisperse samples. Such parallel-conduit systems are fundamentally combined duplicated forms of the '508 apparatus, modified to appropriately handle the multiple pulse-data streams.
Of interest are the functional characteristics of Coulter volumeter conduits such as 10 in FIG. 1 and the prior art which has been developed to facilitate application of such conduits in practical instruments for particle characterization. In the related patent application the characteristics, limitations, and facilitating art of the Coulter conduit have been given a comprehensive review, which is herein incorporated in its entirety by reference. Significant conduit characteristics include: 1) Those defining the conduit sensitive zone Z; 2) Those explaining anomalous pulses generated by particles transiting sensitive zone Z on wall trajectories; and 3) Those explaining extraneous pulses generated by exiting particles recirculating into the exit ambit of sensitive zone Z. For convenience, relevant points of specific interest to the present application may be summarized from said review as follows:
1. As shown in FIG. 1, the partice-sensitive zone Z functionally includes not only the geometric volumeter conduit 10 defined by wall 30 but also the two semielliptical ambit electric fields 31 and 32 coaxial with, and outside the opposing ends of, the geometric conduit; the scale of these ambit fields depends only on the diameter D of the respective entrance and exit orifices, 33 and 34. In addition to producing current pulsations as they transit the geometric conduit, particles may also produce current pulsations if they pass through that portion of the suspending liquid containing the ambit fields. Consequently, the semielliptical equipotentials corresponding to the desired detectability threshold determine the effective spatial extent of the ambit fields 31 and 32. It has been demonstrated that the effective ambit feeds extend outward from the respective entrance and exit orifices 33 and 34 of volumeter conduit 10 approximately one conduit diameter D, with lateral intercepts at 1.15D, if pulse amplitudes from peripheral passages are to be limited to one percent of the theoretical maximum signal-pulse amplitude. For these one-percent equipotentials 35 and 36, the axial length of sensitive zone Z is (L+2D), and for UD=1.2 more than 85 percent of the particle-sensitive zone can be shown to be external to the geometric Coulter conduit 10. The spatial extent of sensitive zone Z increases the likelihood of particle coincidence, requiring greater sample dilution and processing times. Particle coincidence degrades count data directly through lost particle pulses. It also degrades volumetric data indirectly through inappropriate inclusion of misshapen pulses in the volumetric distribution. Adaptive dilution may acceptably limit coincidence artifact (U.S. Pat. No. 3,979,669 to T. J. Godin), or adaptive extension of the counting period may acceptably compensate it (U.S. Pat. No. 4,009,443 to Wallace H. Coulter et al.); but the resulting variable processing times are undesirable in many applications. In principle, the pulse loss due to coincidence can be predicted statistically, and many postcollection corrective techniques have been described in the scientific and patent literature; see, e.g., U.S. Pat. No. 3,949,197 to H. Bader for a review and example. Other approaches estimate pulse loss based on pulse occurrence rate, count, or duration, e.g., U.S. Pat. No. 3,790,883 to P. Bergegere; U.S. Pat. Nos. 3,936,739 and 3,940,691 to W. R. Hogg; U.S. Pat. No. 3,949,198 to Wallace H. Coulter and W. R. Hogg; and U.S. Pat. No. 3,987,391 to W. R. Hogg. Limitations of several are discussed in U.S. Pat. No. 4,510,438 to R. Auer, which proposes correction for the actual coincidence rate as determined by an independent optical sensing modality. These methods may acceptably correct count data for coincidence pulse loss when automated for specific applications, but only those which inhibit incorporation of misshapen pulses can improve the population volumetric distribution.
In addition to coincidence effects, the spatial extent of sensitive zone Z limits pulse signal-to-noise ratios, and therefore particle detectability, in two ways. First, particle contrasts and so pulse amplitudes are limited, since the volumetric sensitivity depends on the ratio of liquid volume displaced by each particle to the volume of liquid in the sensitive zone; and secondly, the noise tending to mask particle contrasts is increased, since it originates thermally throughout this latter volume. Noise originates by two mechanisms, heating noise resulting from dissipation of the excitation current in the resistance of the particle-sensitive zone, and Johnson noise generated in this resistance. These limit the maximum practicable excitation current, on the one hand, and fundamental particle detectability on the other. In the prior art, heating noise has been reduced by providing thermally conductive paths leading away from conduit 10. U.S. Pat. No. 3,361,965 to Wallce H. Coulter and Joseph R. Coulter, Jr., describes one such structure, in which one electrode is formed as a plated metallic coating on the outer surface of a tube closed except for conduit 10 in wafer W. In U.S. Pat. No. 3,714,565 to Wallace H. Coulter and W. R. Hogg the electrical path length through the suspending liquid, and so the thermal noise, is reduced by replacing the second electrode with a metallic element either composing, or coated onto the inner surface of, the tube wall. The thermal effects are described more fully in U.S. Pat. No. 3,771,058 to W. R. Hogg; here, volumeter conduit 10 is formed in a wafer of thermally conductive dielectric and thermally connected to remote cooler regions via electrically and thermally conductive metallic coatings extending onto both planar surfaces of the conduit wafer. In U.S. Pat. No. 4,760,328 the same geometry is described in a structure which integrates sensing electronics onto the sapphire wafer. In all four of these patents the conductors cover extensive areas of the structure and variously approach volumeter conduit 10, but do not extend so close to the conduit as to interact with the effective ambit fields of its particle-sensitive zone Z. However, in U.S. Pat. No. 3,924,180 the Coulter conduit structure is modified by incorporation of thin conductors into the conduit structure, contiguous to conduit orifices 33 and 34, so forming potential-sensing electrodes in a dielectric sandwich through which the conduit penetrates; the intent is to minimize noise contributions to the sensed particle signal from the liquid outside the conduit ambits 31 and 32. Other techniques attempt to minimize noise effects, as for example the noise discriminator described in U.S. Pat. No. 3,781,674 to W. A. Claps, or the averaging of signals from tandem conduit/electrode structures similar to those of aforementioned U.S. Pat. No. 3,924,180 to reduce Johnson noise as described in U.S. Pat. No. 4,438,390 to W. R. Hogg. In critical applications, certain of these may reduce heating noise generated in the conduit, but none significantly improves the volumetric sensitivity. U.S. Pat. Nos. 3,924,180 and 4,438,390 are the subject of further discussion, to follow.
In principle, shorter conduit lengths L can decrease particle coincidence, increase conduit volumetric sensitivity, and decrease thermal noise; in practice, the benefits of decreasing conduit length are limited because, as L approaches zero, sensitive zone Z collapses to the ambit ellipsoid with volume determined by the conduit diameter and the desired threshold of pulse detectability. As indicated in discussion to follow, axial homogeneity of the electric field inside the geometric portion of sensitive zone Z also decreases with decreasing L, and the amplitude of pulses generated by particles transiting conduit 10 correspondingly decreases, with attendant reduction in pulse signal-to-noise ratio.
2. In volumetric applications of Coulter volumeter conduits, the most significant hydrodynamic effects are those on particle trajectory, shape, and orientation during passage through sensitive zone Z. In response to the driving pressure gradient, particles P in the sample compartment are entrained in a developing concentric flow and accelerated toward the entry orifice 33 of volumeter conduit 10. At the entry orifice 33 in FIG. 1, the velocity profile of the constricting flow is quasi-uniform and of a magnitude determined by the desired sample volume, the time allowed to process it, and the cross-sectional area of the conduit The flow just inside the conduit includes a shear layer at conduit wall 30, and particularly for L/D ratios less than about 3.0, the flow profile depends on the edge sharpness of entry orifice 33 and on how closely the kinematic viscosity of the suspending liquid permits it to follow the orifice geometry (for practical reasons, orifice edges are usually sharp, and the shear layer surrounding the developing laminar flow may thicken to appreciably constrict the apparent flow cross-section). When curvature of the edge at orifice 33 is sufficiently gradual, viscosity causes a transition from a quasi-uniform velocity profile toward the parabolic velocity profile of laminar flow. As has been noted, the sensitive zone Z extends outward about one conduit diameter D from the entry orifice 33 in FIG. 1 and is thus overlapped by the convergent flow into conduit 10. The electric field forming that portion of particle-sensitive zone Z within the geometric volumeter conduit 10 is inhomogeneous, only approaching homogeneity at the conduit midpoint for L/D ratios of 2.0 or greater. As a result of the midpoint field inhomogeneity, particles transiting conduit 10 along axial trajectories fail to generate fully-developed pulse amplitudes for conduits with L/D ratios less than 2.5; in addition, particles with similar contrasts generate pulse amplitudes depending on the radial position of the particle trajectory, particularly near wall 30, regardless of the L/D ratio of the conduit As they enter the entry ambit 31 of the sensitive zone, particles on near-axial trajectories (e.g., trajectory A.sub.T) may be deformed by the pressure field, and non-spherical particles will be oriented with their long dimension parallel to flow; such particles generate Gaussian-shaped pulses. Particles entering the sensitive zone outside an axial cone approximately 50 degrees in half-angle will, in addition, be accelerated around the edge at orifice 33 and through the conduit in the annulus near wall 30 containing intense orifice gradients. These orifice gradients cause particles on trajectories such as B.sub.T in FIG. 1 to generate M-shaped pulses of anomalous amplitude and duration due to gradients in, respectively, conduit field and liquid flow. Particles on an intermediate trajectory such as C.sub.T may generate asymmetric pulses, which demonstrate anomalous amplitude only on their leading edge. Due to their anomalous pulse amplitudes, particles following near-wall trajectories (e.g., B.sub.T and C.sub.T in FIG. 1) introduce artifactual high-volume skewness into the sample volumetric distribution, so degrading system ability to resolve particles of nearly identical volumes. The frequency of such pulses depends on the average radial position of the modal trajectories, which in turn is determined by the length L of the conduit, and the portion of the conduit cross section occupied by the orifice gradients. This region, from conduit wall 30 inward to a radius r=0.75(D/2) for typical Coulter volumeter conduits, also defines the maximum particle diameter for which linear volumetric response is obtained. Conduits with L/D=3.3 have been shown to reduce skewness inaccuracies; then, exit modal trajectories are centered inside r=0.66(D/2).
The characteristic M-shaped pulses produced by particles transiting volumeter conduit 10 on trajectories such as B.sub.T or C.sub.T introduce artifacts into the volumetric distribution, the importance of which is attested by the large amount of remedial prior art addressing them. This art is divided between two approaches, the early post-collection one of excluding M-shaped pulses from the processed data and the later direct one of hydrodynamically controlling presentation of particles to the sensitive zone of the conduit. Deletion of such pulses from the volumetric distribution data is suggested in U.S. Pat. No. 3,668,531 to W. R. Hogg, from which FIG. 1 is adapted. Other approaches have been described (U.S. Pat. Nos. 3,700,867 and 3,701,029 to W. R. Hogg; U.S. Pat. Nos. 3,710,263 and 3,710,264 to E. N. Doty and W. R. Hogg; U.S. Pat. No. 3,783,391 to W. R. Hogg and Wallace H. Coulter; U.S. Pat. No. 3,863,160 to E. N. Doty; and U.S. Pat. No. 3,961,249 to Wallce H. Coulter), all of which incorporate gating circuitry responsive to various anomalous parameters of the misshapen pulses by which these pulses may be deleted from the pulse train processed for population distributions. Some of these are discussed in U.S. Pat. No. 3,863,159 to Wallace H. Coulter and E. N. Doty and in U.S. Pat. No. 4,797,624 to H. J. Dunstan et al., either of which well illustrates such gating methods. Gating may also be done in response to a detection signal from an auxiliary electrode (U.S. Pat. No. 4,161,690). The complexity of working implementations encouraged other approaches, and a simple flow-aligning device in front of the Coulter conduit was shown to improve volumetric accuracy (U.S. Pat. Nos. 3,739,268; 4,290,011; and 4,434,398). Further improvement was gained by injecting the particle stream directly into the conduit through an auxiliary flow director (U.S. Pat. No. 3,793,587 to R. Thom and J. Schulz and U.S. Pat. No. 3,810,010 to R. Thom), a technique now known as hydrodynamically focused flow. If the particle suspension is introduced through the flow director while additional liquid medium is appropriately metered through a port in the entry compartment of the dual-compartment vessel, the particles entering this compartment will be entrained into a sheath of the liquid medium and carried through conduit 10 in the core of the composite flow pattern, with two important consequences. Firstly, the directed flow pattern prevents particles entering conduit 10 on trajectories such as B.sub.T and C.sub.T in FIG. 1, thereby eliminating M-shaped pulses. Secondly, all particles transit conduit 10 inside the sheath liquid, which serves to center the particle trajectories inside the cross section of conduit 10 having relatively homogeneous electric fields, further reducing occurrence of anomalous particle pulses. Numerous conduit subassemblies incorporating focused flow have been described (e.g., U.S. Pat. No. 4,014,611 to R. O. Simpson and T. J. Godin; U.S. Pat. No. 4,395,676 to J. D. Hollinger and W. R. Hogg; U.S. Pat. No. 4,484,134 to M. T. Halloran; U.S. Pat. No. 4,515,274 to J. D. Hollinger and R. I. Pedroso; and U.S. Pat. No. 4,525,666 to M. R. Groves; U.S. Pat. Nos. 3,871,770; 4,165,484; 4,253,058; 4,760,328; 5,150,037; and 5,623,200), some of which also include provisions addressing recirculating particles and the best of which can yield nearly ideal volumetric distributions. All add complexity to practical apparatus, and the large fluid volumes required for effective sample focusing make it impractical to volumetrically determine particle concentration by positive-displacement methods. Because the entraining sheath flow restricts the sample stream to a small central portion of the geometric volumeter conduit, a functional concentration of particles within this volume occurs and limitation of coincidence effects typically requires use of lower particle concentrations than with unfocused systems.
3. Regardless of the sharpness of the edge of the entry orifice 33 in FIG. 1, flow at the exit orifice 34 is jetting flow, with a toroidal low-pressure region surrounding the jet and overlapping the exit ambit field 32. For particles exiting orifice 34, the modal trajectories occur in annuli centered at radii r=0.82(D/2) or 0.76(D/2) for conduits with L/D=0.75 or 1.20, respectively, and significant numbers of particles transit the conduit outside r=0.75(D/2), through the orifice gradients of the sensitive zone. The combination of a sharp edge at orifice 33 and the low L/D ratios of typical volunteer conduits also minimizes the stabilizing effect of viscosity, and as a consequence, both the through-flow and jetting patterns are sensitive to imperfections in the edge of entry orifice 33. Conduit L/D ratios of 2.0 or greater result in both smoother flow through the geometric volumeter conduit and less turbulence in the jetting zone outside the exit orifice; exit modal trajectories for such conduits are centered inside r=0.725(D/2). Decelerating particles that have exited the geometric volumeter conduit 10 may be drawn back into the exit ambit 32 (e.g., trajectory D.sub.T in FIG. 1) as the suspending liquid recirculates into the toroidal low-pressure region surrounding the exit jet; if so, they generate extraneous pulses of low amplitude and long duration. Recirculation trajectories also have adverse consequences significant in many applications of the Coulter principle. In contrast to an ideal volumetric distribution, the recirculating particles (e.g., trajectory D.sub.T in FIG. 1) result in a secondary low-volume distribution in the actual sample distribution; this spurious distribution reduces dynamic volumetric range and, for polydisperse samples, may altogether preclude analysis of the smaller particles.
At cost of reduced sample throughput, recirculation pulses may be excluded by pulse gating, either through analysis of pulses from the standard conduit or in response to a thin auxiliary detection electrode located in the conduit's geometric cylinder (aforementioned U.S. Pat. No. 4,161,690). Longitudinal conduit profiles can mechanically reduce the liquid volume available to such particles and may be beneficial in some applications, as noted in U.S. Pat. No. 3,628,140 to W. R. Hogg and Wallace H. Coulter. Other applications are more critical, and many subassemblies incorporating the volumeter conduit have been described which attempt to prevent particles from recirculating into the conduit ambit fields. These either structure the exit flow path so that particles are mechanically prevented from re-entering the sensitive zone (U.S. Pat. Nos. 3,299,354 and 3,746,976 to W. R. Hogg or U.S. Pat. No. 4,484,134 to M. T. Halloran), use auxiliary fluidic circuits to dynamically sweep exiting particles away from the exit orifice (U.S. Pat. No. 4,014,611 to R. O. Simpson and T. J. Godin) or combine these two approaches (U.S. Pat. No. 3,902,115 to W. R. Hogg et al. and U.S. Pat. No. 4,491,786 to T. J. Godin, which contains a review of such methods). Several other implementations have also been described (U.S. Pat. Nos. 4,253,058; 4,290,011; 4,434,398; 4,710,021; 5,402,062; 5,432,992; and 5,623,200). The dynamic sweep-flow method is widely used and involves metering appropriate volumes of the liquid medium through a second inlet port in the exit compartment of the dual-compartment vessel, whereby the particles exiting conduit 10 are swept out of exit ambit field 32. These complex subassemblies can essentially eliminate recirculating particles, may include shaped conduits, and often include additional structure addressing effects of particles following wall trajectories. However, all add complexity to practical apparatus, and the large fluid volumes required for effective sweep-flow make it impractical to volumetrically determine particle concentration by positive disablement methods. An approach due to M. T. Halloran (in aforementioned U.S. Pat. No. 4,484,134) potentially avoids need for auxiliary fluidic circuits and structures, by extending the insulating discs of aforementioned U.S. Pat. No. 3,924,180; of an inner diameter substantially equal to that of the volumeter conduit, such extensions mechanically prevent recirculation of exiting particles into the exit ambit of the conduit and when carefully constructed can provide hydrodynamic advantages of long conduits. However, for many applications of the Coulter principle such structures require complex mechanical designs difficult to implement and which tend to clog in use.
In the invention of the related application, electric and hydrodynamic fields in the vicinity of the two-terminal volumeter conduit are directly amended, whereby the need for dynamic sweep flow is avoided and the need for hydrodynamically focused low is minimized. In addition the coincidence volume of the said field-amending conduit is about 30 percent of that of the Coulter conduit used in the apparatus of the '508 and '328 patents, for geometric conduits of the same configuration and dimensions. Consequently, many of the inaccuracies encountered with the prior-art apparatus is avoided, so eliminating much complexity in apparatus make up. Prior-art two-terminal apparatus of the '508 type, containing none of the aforementioned facilitating art, provided substantially ideal volumetric measurements when the new field-amending volunteer assembly was substituted for the wafer W comprising the FIG. 1 conduit.
Maximum particle-pulse amplitudes are obtained with a given Coulter volumeter conduit when used with the greatest excitation current compatible with generation of acceptable thermal artifacts in the liquid column within the conduit, but practicable current densities may be limited in biological applications to ones incapable of causing breakdown of cell membranes. To minimize artifacts due to electrochemical effects such as polarization and generation of gas bubbles, large-area electrodes located well away from the sensitive zone Z of conduit 10 are preferred. The capacitance of typical electrodes acts to limit rise times and fall times of potential changes between the electrodes, whether these be due to particles transiting the sensitive zone of the conduit or ramping the aperture current so that cellular internal conductivity may be determined, viz., U.S. Pat. No. 4,220,916. Although some two-terminal conduit structures incorporated comparatively small electrodes (e.g., U.S. Pat. No. 4,157,498; aforementioned U.S. Pat. No. 3,361,965 to Wallace H. Coulter and Joseph R. Coulter, Jr.; U.S. Pat. No.3,714,565 to Wallace H. Coulter and W. R. Hogg; U.S. Pat. No. 3,771,058 to W. R. Hogg; or U.S. Pat. No. 4,760,328), these electrodes only approached the volumeter conduit and did not extend into the ambit electric fields 31 or 32 of its sensitive zone, to limit electrochemical artifacts. However, aforementioned U.S. Pat. Nos. 4,290,011 and 4,434,398 describe an elongate conduit which incorporates at its entry orifice one of the two excitation/sensing electrodes as a ring encircling, and in electrical contact with, the conduit flow stream. In addition, U.S. Pat. No. 4,224,567 and aforementioned U.S. Pat. No. 4,484,134 describe similar elongate structures incorporating two thin excitation/sensing electrodes, each surrounding the suspension stream at an orifice of the functional Coulter conduit. (As indicated in the latter patent, control of electrochemical artifacts may require use of auxiliary excitation electrodes, located in remote vessels and connected to the orifice electrodes via channels filled with suspending medium.) These structures are complex, tend to clog, and have received little application.
Substitution of a conduit of different geometry or changes in resistivity of the suspending medium may affect the excitation current density in the conduit; in U.S. Pat. No. 3,944,917 to W. R. Hogg et al. a set of auxiliary sensing electrodes, located in the suspending medium near the conduit orifices, provides a control signal which enables compensation of the excitation current applied to the excitation electrodes for resultant changes in resistance of the liquid column within the conduit. Controlling the diversion of excitation current through the fluidic subsystem requires a means of isolating the conduit from the remainder of this system, and other four-terminal art can achieve this through active means, by which auxiliary control electrodes are driven by a bootstrap circuit, viz., U.S. Pat. No. 4,972,137 to H. J. Dunstan and I. D. Gilbert. Additional examples are known of similar four-terminal art, but all suffer more or less indeterminate electrode placement relative to the conduit. In some desirable applications of such art the result is an unacceptable degradation in the dynamic characteristics of the operational mechanism.
In the 1970s it was recognized that if no current were passed through independent electrodes (i.e., the electrodes are potential sensing rather than current sensing) such electrodes could then be both compactly designed and advantageously fixed near the conduit orifices, thereby reducing electrode capacitance, pulse rise times, and locational uncertainty. Such four-terminal sensing art developed around two distinct approaches, one by Leif and another by Salzman, both using high-impedance sensing circuits. Both approaches also contain the volumeter conduit within a stacked five-layer assembly, each of the four outer layers including an opening coaxially aligned with the functional Coulter conduit through the central layer. However, functional properties of the two resulting sensitive zones differ appreciably, due to structural differences in the two five-layer assemblies.
In the Leif structure, potential-sensing electrodes are located near, but not in, the sensitive zone of a conventional Coulter conduit. The most relevant example is described by R. A. Thomas, B. F. Cameron, and R. C. Leif [Computer-based electronic cell volume analysis with the AMAC II transducer, The Journal of Histochemistry and Cytochemistry, 22:626-641, 19741]. This example (12 in FIG. 2) comprises thick platinum potential-sensing electrodes 13 and 14, each separated from a conventional Coulter conduit wafer W by a circular insulating spacing-element 23 or 24. The individual components 13, 14, 23, and 24 are several tens of times the diameter of volumeter conduit 10' (D=0.100 mm, L/D=1.0), and each contains a central circular opening 25, 28, 26, or 27, respectively, of diameter 0.010" (0.254 mm or approximately 2.5D) aligned coaxially with the smaller Coulter conduit 10' during assembly. The thickness of each electrode 13 and 14 and each spacing-element 23 and 24 is also 0.010". In use, conduit 10' and combined openings 25/26 and 27128 in each electrode/spacing-element couple 13/23 and 24/14, respectively, form a fluid-filled channel interconnecting the two compartments of the dielectric vessel containing the suspending liquid in which excitation electrodes are immersed. Sensing electrodes 13 and 14 are in electrical contact with the suspending medium carrying the particles through said channel and are operatively connected to high-impedance sensing circuits, so that no significant current flows between them.
As for the conventional Coulter conduit 10 in FIG. 1, characteristics of the electric and hydrodynamic fields within volunteer conduit 10' of FIG. 2 Leif structure 12 are co-determined, and within the fluid-filled channel surrounded by wall 30 the electric flow established by the current between the excitation electrodes is inhomogeneous. As indicated in FIG. 2, in and near Coulter conduit 10' the field distribution is similar to that for conduit 10 of the Coulter wafer W used conventionally, as in FIG. 1. Particles P carried through the FIG. 2 sensitive zone Z.sub.L by coaxial hydrodynamically focused flow (e.g., on trajectory A.sub.T ) produce particle pulses having improved rise times when sensed between electrodes 13 and 14. When the flow director is positioned off the conduit axis so that particles transit conduit 10' on off-axis trajectories such as B.sub.T ', anomalous pulses result similar to those seen for particles on trajectory B.sub.T in FIG. 1; the frequency of such pulses (and therefore the severity of resultant skewness the volumetric histogram) increases as the flow director is positioned to cause near-wall trajectories, thus demonstrating orifice and wall gradients in the Leif structure comparable to those encountered when Coulter wafer W is used conventionally.
As also indicated in FIG. 2, the one-percent equipotentials 35' and 36' of the ambit fields delimiting sensitive zone Z.sub.L do not directly approach sensing electrodes 13 and 14. In comparison with sensitive zone Z in FIG. 1, the primary coincidence volume contained between the one-percent equipotentials 35' and 36' is of similar axial extent, but somewhat flattened and curtailed laterally by the inner wall of openings 26 and 27. Consequently, the primary coincidence volume of Coulter wafer W when used in Leif structure 12 is somewhat smaller than when used conventionally. However, while the field distribution in and near Coulter conduit 10' in FIG. 2 is similar to that for the conventional Coulter wafer W of FIG. 1, it is superimposed on a lower-gradient field distribution (not shown) due to the impedance of the liquid columns within openings 26 and 27 in spacing-elements 23 and 24. Therefore, both the excitation electrodes and the sensing electrodes 13 and 14 respond to impedance changes in the liquid columns within these openings as well as in sensitive zone Z.sub.L, and pulses generated by single particles consist of a substantially conventional pulse generated during passage through sensitive zone Z.sub.L. However, such particle pulses are superimposed on a low-amplitude pedestal originating in the field gradient along these fluid columns in openings 26 and 27. If two particles simultaneously occupy positions within any responsive portion of the total fluid channel, the resultant pulse amplitude will be artifactual for both particles. Consequently, conduit 10' of Leif structure 12 has a secondary coincidence volume which does not exist for the FIG. 1 Coulter volumeter conduit 10, and this secondary coincidence volume is considerably larger than the primary coincidence volume, particularly when particle pulses are sensed between the excitation electrodes. The compound nature of the individual particle pulses generated by Leif structure 12 and the attendant secondary coincidence volume both tend to complicate analysis of the particle pulses.
In operation, convergent fluidic flow begins developing outside the entry to opening 25 in electrode 13 of Leif structure 12, with a further convergent transition at the entry to conduit 10'. Thus, the combined thicknesses of electrode 13 and spacing-element 23 act to straighten trajectories of particles entering the sensitive zone Z.sub.L so that the frequency of particles on trajectories such as B.sub.T or (particularly) C.sub.T in FIG. 1 is reduced, together with the number of resulting anomalous particle pulses. However, a significant number of particles may still transit the FIG. 2 conduit 10' on near-wall trajectories similar to B.sub.T ', with resultant anomalous pulses, unless hydrodynamically focused-flow methods are used.
Thicknesses of insulating spacing-element 24 and electrode 14 in Leif structure 12 similarly reduce availability of the sensitive zone to particles on recirculating trajectories such as D.sub.T in FIG. 1, thereby substantially preventing extraneous particle pulses. However, suspension flow through conduit 10' of Leif structure 12 also retains characteristics of flow through conventional Coulter conduit 10 in FIG. 1, including sensitivity to orifice edge defects. Construction of Leif structure 12 to working precision is sufficiently difficult that in later work the design was simplified, by replacing circular platinum electrodes 13 and 14 of FIG. 2 with platinum pins fixed in insulating spacing-elements 23 and 24.
In the Salzman structure, the volumeter conduit passes through potential-sensing electrodes so that the sensing electrodes are located within the conventional volumeter conduit [G. C. Salzman, P. F. Mullaney, and J. R. Coulter; A Coulter volume spectrometer employing a potential sensing technique, Biophysical Society Abstracts, 17th Annual Meeting, Abstract FPM-F11; Biophysical Joumal, 13:302a, 1973]. The only known description appears in aforementioned U.S. Pat. No. 3,924,180 to G. C. Salzman, J. R. Coulter, P. F. Mullaney, and R. D. Hiebert. The objects of the '180 patent were reduction of sensitive-zone coincidence volume and particle pulse-width, with improvement of pulse signal-to-noise ratio. In contrast to the FIG. 2 Leif structure 12, the Salzman structure (38 in FIG. 3) places thin platinum potential-sensing electrodes 41 and 42 in contact with functional Coulter wafer W', with each electrode isolated from the suspending liquid by a circular insulating element 43 or 44. As for the Leif structure, the individual sensing electrodes 41 and 42 and insulating elements 43 and 44 in the Salzman structure each contain a central circular opening (46, 47, 45, or 48, respectively) aligned coaxially with Coulter conduit 10"; however, in the Salzman structure 38 these openings are of the same nominal diameter as Coulter conduit 10". The openings through insulating element 43, electrode 41, Coulter wafer W, electrode 42, and insulating element 44 of the Salzman structure collectively form a continuous wall 30' defining volumeter conduit 40 of uniform diameter and interconnecting the two compartments of the vessel containing the suspending liquid in which excitation electrodes are immersed. The walls of openings 46 and 47 in sensing electrodes 41 and 42 are in electrical contact with the suspending medium carrying the particles through conduit 40, and electrodes 41 and 42 are operatively connected to high-impedance sensing circuits, so that no significant electrical current flows between them.
For the Salzman structure 38 of FIG. 3, characteristics of the hydrodynamic and electric fields are co-determined and theoretically identical to those for a Coulter conduit of length L', with one-percent equipotentials 35 and 36 in the latter when particles are sensed between the excitation electrodes. However, in the Salzman four-terminal approach it is intended that only that portion 10", of conduit 40 between sensing electrodes 41 and 42 produce a signal pulse on passage of a particle through the conduit. Due to the longer total conduit 40 the electric field in 10" is substantially homogeneous, being isolated from the orifice field gradients by the thickness of insulating elements 43 and 44. Because the separation between electrodes L (=0.144 mm), conduit length L' (=0.412 mm), and conduit diameter D (=0.087 mm) were important to the objects of the invention, these dimensions were specified in the '180 patent. However, neither the thicknesses nor the lateral extents of electrodes 41 and 42 and insulating elements 43 and 44 are related to the inventive objects, and none of these dimensions were addressed. Thus, in FIG. 3 of the '180 patent, insulating elements 43 and 44 are indicated to be identical to the functional Coulter wafer W', i.e., 0.144 mm thick for the example. Because the collective thicknesses of elements 43, 44, and W' then exceed the stated length L' of the conduit, electrodes 41 and 42 must be vanishingly thin, i.e., not a small fraction of a conduit diameter in thickness as illustrated in the present FIG. 3. Thin electrodes 41 and 42 thus coincide with equipotentials of the homogeneous electric field, viz., the electric field within the conduit is unaffected by the presence of the electrodes if the latter are connected to high-impedance sensing circuits. Thus, the extent of sensitive zone Z.sub.S is substantially defined by the thickness L of wafer W' separating electrodes 41 and 42. By decreasing the thickness L of functional Coulter wafer W' the coincidence volume of sensitive zone Z.sub.S may in principle be decreased, with correspondingly decreased particle pulse-width; insulating elements 43 and 44 serve to isolate sensing electrodes 41 and 42 (due to the fluid resistances within openings 45 and 48 therein) from noise originating in the suspending medium within the dual-compartment vessel. For sufficient thicknesses of elements 43 and 44, there are no substantial regions of high electric-field gradients near sensitive zone Z.sub.S to cause either anomalous or extraneous pulses.
In FIG. 3, the hydrodynamic field begins developing convergent flow near the entry to opening 45 in insulating element 43. If of sufficient combined thickness, electrode 41 and insulating element 43 permit trajectories of particles to be straightened, so that particle trajectories through sensitive zone Z.sub.S such as B.sub.T or C.sub.T in FIG. 1 are essentially eliminated. Sufficient combined thickness of electrode 42 and insulating element 44 similarly precludes availability of sensitive zone Z.sub.S to particles on recirculating trajectories such as D.sub.T in FIG. 1, thereby eliminating extraneous particle pulses. However, these hydrodynamic effects are less important than for the Coulter conduits 10 or 10' of FIGS. 1 or 2, since there are no significant electric-field gradients near electrodes 41 and 42. Consequently, particles on near-wall trajectories through sensitive zone Z.sub.S of FIG. 3 do not produce anomalous pulses when the particle pulse is sensed between electrodes 41 and 42. However, suspension flow through conduit portion 10" of the Salzman structure 38 retains characteristics of flow through conventional Coulter conduit 10 in FIG. 1, including sensitivity to defects in wall 30'. Construction of Salzman conduit structures to the requisite quality has also proven difficult.
Both the Leif and Salzman potential-sensing approaches introduce liquid columns of comparatively small diameter between the excitation electrodes and the sensitive zone of the conduit structures (liquid-filled openings in spacing-elements 23 and 24 in FIG. 2 or elements 43 and 44 in FIG. 3, respectively). Electrically in series with the volumeter conduit, these liquid columns not only isolate the sensitive zone from electrical noise coupled into the bulk of the suspending medium in the two compartments of the dielectric vessel, but form part of a voltage divider between the excitation electrodes. The portion of the total pulse signal developed between the sensing electrodes on passage of a particle is determined by the ratio of the change in impedance between the sensing electrodes to the total impedance existing between the excitation electrodes. Because the liquid columns separate the sensitive zone Z.sub.L from the two sensing electrodes 13 and 14 in the Leif structure of FIG. 2, particle pulses are superimposed on a low-voltage pedestal originating in the potential gradient along the column, and pulse processing may be complicated in some analytic methods, as has been noted. In the Salzman structure of FIG. 3, this disadvantage is avoided because sensing electrodes 41 and 42 are adjacent to sensitive zone Z.sub.S, but at the expense of reduced particle-pulse amplitude between the two sensing electrodes. The reduced pulse amplitude is due to the voltage-divider effect of the small-diameter liquid columns through elements 43 and 44, by which Z.sub.S is electrically connected to the bulk of the suspending medium in the dual-compartment vessel.
In the '180 patent there is suggested, but not described in detail, a variant structure obtained through omission of both insulating elements 43 and 44 in FIG. 3; as shown in FIG. 4, uninsulated electrodes 41' and 42' of this variant structure are exposed to the suspending liquid on their lateral faces as well as on the wall of openings 46' or 47' through them. Omission of elements 43 and 44 does eliminate the voltage-divider effect due to the fluid columns in the openings therein, but also eliminates a stated goal of the '180 patent, the beneficial hydrodynamic effects of the long FIG. 3 conduit 40. The hydrodynamic field for this variant four-terminal structure is theoretically identical to that of a FIG. 1 two-terminal Coulter volumeter conduit of equivalent conduit dimensions, and its axial electric-field distribution is also similar to that of the conventional conduit. However, as illustrated in FIG. 4, the thin sensing electrodes on the lateral surfaces of functional Coulter wafer W' impose new equipotentials on the distribution of the electric field near W' and near the wall of conduit 40', with some reduction in the effective spatial extent of the electric field near W'. Thus, the one-percent equipotentials 35" and 36" of the FIG. 4 electric field distribution originate at the openings of conduit 40', rather than at a distance from these as for functional conduits 10 or 10' in the volumeter structures of FIGS. 1 or 2, respectively.
For particle pulses sensed between the excitation electrodes, particles on trajectories such as A.sub.T, A.sub.T, C.sub.T, or D.sub.T generate pulses similar to those on similar trajectories through the FIG. 1 conduit, except that in the FIG. 4 conduit the latter trajectory can result in pulses of greater amplitude and shorter duration. For pulses sensed between electrodes 41' and 42', the pulse effects of electric-field gradients outside openings 46' and 47' therein are significantly reduced, and the anomalous or extraneous pulses generated by particles on trajectories B.sub.T and C.sub.T or D.sub.T, respectively, are substantially eliminated. Suspension hydrodynamics through the FIG. 4 structure share with the FIG. 1 and FIG. 2 structures a sensitivity to imperfections in the entry region; further, due to contiguity of sensing electrodes 41' and 42' to the conduit orifices, the electric field distribution is significantly more sensitive to such imperfections in the variant Salzman structure than for either of the latter structures.
Structure 38 of FIG. 3 is illustrated in U.S. Pat. No. 4,019,134 to W. R. Hogg, as suited to providing a control signal responsive to changes in conductivity of the suspending liquid; and in aforementioned U.S. Pat. No. 4,224,567 it is specified as suited to resistance calibration of the primary apparatus. A derivative structure taught by M. T. Halloran (aforementioned U.S. Pat. No. 4,484,134) is yet another implementation; in it dielectric tubes similar to those in aforementioned U.S. Pat. Nos. 4,290,011 and 4,434,398 are used as elements 43 and 44 in the FIG. 3 structure. In aforementioned U.S. Pat. No. 4,161,690 a form of the Salzman structure is used to allow assessment of the time course of primary pulse development as sensed between the two-terminal excitation/sensing electrodes, viz., at least one auxiliary sensing circuit, connected between one of the excitation/sensing electrodes and a dedicated auxiliary electrode contacting the fluid within the conduit channel, provided an auxiliary signal permitting either selecting primary pulses having certain characteristics or sampling primary pulses at a desired stage in their development. In this latter patent, one insulating element (43 or 44) and one electrode (41 or 42) are omitted from the present FIG. 3 structure 38 to provide the preferred implementation.
Certain applications may benefit from use of sequential conduits, so that the same particle may be either passively sensed more than once (aforementioned U.S. Pat. No. 3,793,587 to R. Thom and J. Schulz) or actively modified in the second conduit by a field-effected operation applied in response to the measurement made in the first (e.g., aforementioned U.S. Pat. No. 4,525,666 to M. R. Groves). Apparatus described in these patents incorporate plural discrete Coulter conduits, each conduit interconnecting sequential compartments containing two-terminal excitation/sensing electrodes immersed in the suspending liquid. For each conduit, electrical circuitry connected between a pair of adjacent electrodes provides excitation current and may derive a particle pulse, as in the =508 apparatus. However, fluidic subsystems substantially more complex than are needed in the '508 apparatus must be used. Further, the unavoidable separation between sequential discrete conduits requires use of additional methods (e.g., such as described in U.S. Pat. No. 4,184,766 to W. R. Hogg) for correlating the activities within the individual conduits; for these, correlation accuracy scales directly with particle size and inversely with conduit separation. Because the latter is grossly indeterminate with respect to particle size, these complex methods seldom achieve high creditability. Due to the jetting flow exiting traditional Coulter conduits, flow patterns in the fluidic gap separating such sequential conduits require hydrodynamically focused flow to maintain the particle stream within a useful portion of the second conduit (e.g., U.S. Pat. No. 3,793,587).
Integrated structures incorporating tandem sensitive zones should offer attractive advantages over the discrete sequential structures of aforementioned U.S. Pat. Nos. 3,793,587 and 4,525,666. In particular, the fluidic gap and its jetting flow would be eliminated, the need for complex fluidic subsystems and methods based on hydrodynamically focused flow would be minimized, and correlation problems experienced with discrete sequential-conduit structures would be significantly reduced by the fixed design of fluidically-closed integrated structures. Both the '180 patent and the aforementioned U.S. Pat. No. 4,161,690 suggested assemblies incorporating additional thin sensing electrodes into integrated structures containing a continuous composite conduit, but neither patent described or claimed such structures. An extended form of the '180 conduit, containing a plurality of functional Coulter conduits in fluidic sequence, was proposed in aforementioned U.S. Pat. No. 4,438,390. The many precision components and precise assembly requirements of such integrated structures make them extremely difficult to build, and few have been reduced to practice. Specifically, prototypes of the '180 structure are known to have been tested, and forms of art in aforementioned U.S. Pat. No. 4,161,690 have been commercialized. Other integrated conduit structures have received little practical use, including those of the '390 patent and the aforementioned U.S. Pat. No. 4,484,134, both having in common the assignee of the related and present applications.
Perhaps more pertinent to this lack of development may be the fact that the prior art regarding integrated structures containing electrodes does not anticipate aspects important to practical realization of these structures. In particular, the prior art only recognizes electrodes in integrated conduit structures as potential-sensing interface devices, whereby the passage of a particle through the volumeter conduit may be detected by the sensing circuitry. This prior art does not recognize any functional effect of the electrodes on the electric fields within conduits of such structures but, through use of thin electrodes, substantially prevents the electrodes from affecting conduit field distributions. Specifically, aforementioned U.S. Pat. Nos. 3,924,180; 4,019,134; 4,161,690; 4,224,567; 4,290,011; 4,438,390; 4,434,398; and 4,484,134 typically illustrate electrode thicknesses to be less than the diameter of the Coulter conduit in thickness, but little discussion is given. As has been noted in discussion of the FIG. 3 Salzman structure 38, the '180 patent describes neither the lateral extent nor the thicknesses of the electrodes, but gives dimensions of the other assembly components which force the thicknesses to be vanishingly thin. The structure of the '180 patent is illustrated, but not further described, in U.S. Pat. No. 4,019,134; and in U.S. Pat. No. 4,224,567 it is specifically cited, but not further described. U.S. Pat. Nos. 4,290,011 and 4,434,398 illustrate an elongate conduit, which incorporates at its entry orifice one of two excitation/sensing electrodes as a ring of unspecified dimensions. In U.S. Pat. No. 4,161,690 the thickness of the internal auxiliary electrode is said to be small in relation to length L of the fluid channel; in the '390 patent the thickness of each electrode is illustrated as being significantly less than Coulter wafer thickness L; and in U.S. Pat. No. 4,484,134 the thickness of each electrode is said to be of the order of 5 percent of the Coulter conduit diameter D. This universal use in the prior art of electrodes thin with respect to conduit dimensions demonstrates the unanticipated and non-obvious nature of the interaction between electrode dimensions and conduit functional characteristics. Because the '390 patent claims conduit structure described or claimed in much of the prior art, it will be taken as representative.
In the '390 patent structure is claimed which is based on at least two Coulter wafers, assembled between at least three sensing electrodes; at least one electrode is sandwiched between two Coulter wafers and the volumeter conduit passes through said sandwich. Each pair of adjacent electrodes is operatively connected to a differential sensing circuit, so that as a given particle transits the plural portions of the composite conduit, multiple particle pulses sequenced in time can be developed.
1. If remote sensing electrodes are used, the conduit structure and symmetric ambit fields correspond to those of FIG. 3 structure 38, with Coulter wafer W' and electrode 42 omitted; Except for the surface constituting the wall of the opening through the electrode, the latter is insulated from the sample suspension by elements 43 or 44. The conduit structure is that of U.S. Pat. No. 4,161,690, in which connections of the sensing circuitry may be different.
2. If the second sensing electrode is located on the outer surface of one of the outer-most Coulter wafers, the conduit structure corresponds to that of FIG. 3 with either insulating element 43 or 44 omitted, and one ambit field resembles those of the FIG. 3 structure 38 while the ambit field at the affected orifice resembles those of the FIG. 4 structure 38'.
3. If the third sensing electrode is located on the outer surface of the second of the outermost Coulter wafers, a tandem version of the FIG. 4 variant Salzman structure 38' is formed, and both ambit fields resemble those shown in FIG. 4.
4. Any of the preceding structures can in principle be extended in an obvious manner, by adding Coulter wafers and/or electrodes in a fluidically continuous manner. Thus, adding a Coulter wafer to insulate the exposed electrode of the second structure results in the '180 patent structure 38 of FIG. 3, while appropriately adding two Coulter wafers and another electrode results in a tandem version of this structure.
Because of their thinness, each sensing electrode essentially coincides with an equipotential in the conduit field distribution. In all '390 structures, the internal ambits between adjacent Coulter conduits resemble those within openings 46 or 47 of electrodes 41 or 42 of the FIG. 3 Salzman structure 38. Consequently, particle pulses from the outer sensitive zones of any '390 structure are both asymmetric and unlike those from any internal sensitive zone. The dissimilar rise and fall times may significantly increase the difficulty of using such pulses, e.g., in the pulse-averaging method taught in the '390 patent. Of lesser importance, but of potential concern in some applications, is the trajectory-dependent difference in asymmetric pulse characteristics generated by particles transiting asymmetric '390 structure, e.g., any '390 structure having an equal number of Coulter wafers and electrodes.
A further limitation becomes apparent when integrated tandem-conduit structures are considered for use in applications requiring active control of conduit fields, e.g., aforementioned U.S. Pat. Nos. 4,525,666 and 4,972,137, both having in common the assignee of the related and present applications. In the first of these patents, remote excitation/sensing electrodes are used to actively modify a biological particle (cell) by ramping the electric field in a second discrete sequential conduit in response to a parameter sensed in a preceding discrete conduit; in the second, remote auxiliary electrodes are used to isolate a single discrete conduit from the rest of the fluidic subsystem. The confined fluid flow through integrated conduit structures would make such active use of incorporated electrodes very advantageous, although such use is unknown in the prior art. However, as illustrated in FIGS. 1 through 4, electric field distributions bulge convexly outward from regions of high field intensity in the sensitive zones into regions of lower field intensity, e.g., into the suspending medium in the compartments interconnected by the volumeter conduit. If a higher field intensity is induced in any selected portion of an integrated conduit structure, e.g., the FIG. 3 conduit 40, by appropriately connecting a second excitation source between any of the sequential pairs of electrodes (viz., the excitation electrode in the vessel external to element 43 and electrode 41, electrodes 41 and 42, or electrode 42 and the excitation electrode in the vessel external to element 44), the resultant high field intensity within said portion will cause the ambit fields of that portion to bulge through the openings 46 or 47 of thin electrode 41 or 42 into the adjacent portion more or less according to the ratio of the field intensities in the two portions. If the second excitation source is made to provide a time varying field within the selected conduit portion, the ambits of said portion will intrude into the adjacent portions in a time-varying manner. Thus, the thin electrodes of prior-art integrated conduit structures do not favor active use of the incorporated electrodes, since the field manipulations required to achieve the desired conditions in one sensitive zone will interact with and affect the electric fields of adjacent sensitive zones. The consequent substantial change in spatial extent of the effective sensitive zone in any adjacent portion of the volunteer conduit will degrade the volumetric accuracy of measurements made therein.
Although the prior art alludes to hydrodynamic advantages of longer conduit structures, it neither details the origin of such advantages nor suggests a specific method whereby such advantages may be systematically obtained. Further, other than nonspecific observations (e.g., in the '390 patent) that the walls of the conduit should be fluidically smooth in integrated conduit structures, the prior art does not recognize any functional effect of the electrodes on hydrodynamic fields within the incorporated conduits. Prior-art conduit structures often practically require hydrodynamically focused flow or sweep flow, and in addition to undesirable complexity, such apparatus precludes positive-displacement determinations of particle concentration.
There is no question that the accuracy, resolution, and convenience of the two-terminal Coulter art have substantially benefited through the teachings of the many patents cited above and in the related application. While the potential-sensing, tandem, and active forms of the Coulter art potentially offer many advantages which may enable new forms and applications of the Coulter principle, prior-art apparatus and methods based on these have demonstrated many practical limitations and disadvantages. Some of the latter originate in the functional properties of the traditional Coulter conduit, for which a rich prior art in facilitating apparatus and methods has developed. As discussed in the related application, it is preferable to obviate such facilitating apparatus and methods, by directly amending field characteristics of the volumeter conduit.
It is desirable that a practicable integrated volumeter structure be provided which offers improved characteristics in its hydrodynamic and electric conduit fields. It is desirable that the improved volumeter structure be useful with apparatus for four-terminal potential sensing methods, e.g., aforementioned U.S. Pat. Nos. 3,924,180; 4,019,134; 4,224,567; or 4,484,134 or bridge methods such as described in the aforementioned publication by R. A. Thomas, B. F. Cameron, and R. C. Leif. It is also desirable that the improved volumeter structure lend itself to use in methods requiring plural tandem volumeter conduits, e.g., those of aforementioned U.S. Pat. Nos. 3,793,587 or 4,438,390. Furthermore, it is desirable that the improved volumeter structure be useful i n methods requiring active control of electrode potentials or electric-field intensity in one or more of plural conduits, e.g., aforementioned U.S. Pat. Nos. 4,525,666 or 4,972,137, respectively.